This project renewal retains an overall aim of enhanced quantitative mechanistic understanding of cardiac myocyte function related to calcium signaling. Specific Aims focus on novel timely topics related to 1) diabetic hyperglycemia, 2) mitochondrial function and 3) local Ca signaling. We recently found that diabetic hyperglycemia induces O-GlcNAcylation of CaMKII at Ser279, causing autonomous CaMKII activation, ryanodine receptor phosphorylation and enhanced arrhythmogenesis. Since CaMKII affect numerous ion channels and transporters, this opens a new area to test how hyperglycemic CaMKII activation alters myocyte Ca handling and electrophysiology (Aim 1). Recent molecular identification of mitochondrial ion and availability of novel fluorescent sensors creates a unique opportunity now to better understand mitochondrial Ca handling related to energetics (Aim 2). Mitochondrial ROS production is increased by extremes of mitochondrial [Ca] and energy demands, and ROS can alter CaMKII activation and ion transporters). Excitation-contraction coupling in heart involves very local Ca signaling directly, but much downstream Ca via calmodulin, calcineurin and CaMKII that effects things like mitochondrial and cell death, altered gene expression in disease, also involves very local signaling that is not understood. We will help to unravel this signaling using novel targeted sensors and optical methods (Aim 3). These complementary aims will enrich our quantitative knowledge of myocyte and mitochondrial Ca signaling and effects of hyperglycemia and ROS. Our three Aims, as hypotheses, are: 1) Hyperglycemia alters myocyte Ca fluxes and ionic currents via O-GlcNAc-CaMKII-dependent effects, 2) Mitochondrial Ca regulation influences myocyte energetics and ROS effects on [Ca]i regulation, and 3) Junctional cleft [Ca] regulates calmodulin, calcineurin and CaMKII differently than does bulk cytosolic [Ca]. These studies will accrue valuable new quantitative mechanistic data, which we will integrate into improved comprehensive understanding of cardiac function.